Development of a Panel for Dengue Virus

Maria Rios, PhD CBER/FDA

Blood Products Advisory Committee MeetingDecember 14, 2010

The Need for Standard Reagents for Dengue virus (DENV)

There are four known serotypes of DENV

Reference reagents are needed to evaluate test performance

Reference reagents require large volumes of the 4 DENV serotypes; clinical specimens are limited in volume and not suitable

Laboratory strains for all 4 serotypes are available for the development of standard reagents

Detection of Active DENV Infection

Two types of assays can detect active early infection :

Nucleic acid testing (NAT) - for the detection of viral RNA which is produced soon after initiation of viral infection much earlier than the antibody production

Antigen assays - Non-structural protein (NS1antigen) known to be produced and secreted in the early stages of infection after appearance of viral RNA and before antibody production

Blood Screening for Viruses

NAT platform for viral RNA detection has been extensively used to screen blood donations

HIV, HCV and WNV are examples of RNA viruses where detection of genetic material is the most sensitive approach to prevent transmission by transfusion

Epidemiological studies have used NAT assays for the detection of DENV among blood donors

FDA is proactively involved in the production of RNA standard reagent for DENV to facilitate development and evaluation of performance of new assays, and future need for lot release

DENV antigen panel will be developed, if needed

Efforts Towards Standards Development

CDC has provided viral isolates from each of the DENV serotypes to be used as standards:

– DENV-1, Hawaii

– DENV-2, New Guinea C

– DENV-3, H-87

– DENV-4, H-241

These are laboratory strains that have undergone multiple propagations after virus isolation from human specimens

from Philippines

DENV RNA Titer Standardization

Lack of consensus for viral titer

– Viral titer has been defined by plaque assay (PFU)

– Number of viral particles per PFU has broad range (1 – 1000 virions)

– Need for correlation of RNA copies with PFU

– Non-infectious particles (defective) may be detected by NAT but not by infectivity assays

Copy number assignment is necessary to define analytical sensitivity

Steps of DENV RNA Standard Preparation

1. 240 mL of virus stocks from each of the 4 serotypes were produced in C6/36 mosquito cells

2. Preliminary characterization of stocks in house

Genetic sequencing

Infectivity titer by plaque assay (PFU and FFU)

Viral RNA concentration determined in PDU (PCR Detectable Units) by limiting dilution TaqMan

3. A portion of the Stocks were heat inactivated (HI) by incubation at 62°C for 1 hour

4. Dilutions of both live and HI stocks were prepared and frozen at -80°C

Standardization of DENV RNA stocks: Preliminary characterization

1. Two concentrations of stocks from each serotype were retested at:

FDA

NIAID/NIH (Drs Eagle and Goncalves)

CDC (Dr Lanciotti)

NYSDoH (Dr Kramer)

Gen-Probe (Dr Linnen)

2. HI was independently confirmed by cultivation at FDA, NIAID/NIH; NYSDoH and CDC laboratories

Standardization of DENV RNA stocks: Preliminary characterization

3. The Stocks concentrations were defined by average of preliminary characterization performed by 5 different laboratories

Viral Load (PDU/ml) Titer (PFU/ml)

DENV-1 1011.7 105

DENV-2 1011.6 105

DENV-3 1010.6 104

DENV-4 1010.5 104

4. Stability studies in pre-characterized stocks are ongoing

Ongoing Stability Studies

For evaluation of the candidate preparation stocks were diluted in:

– VP-SFM – tissue culture medium

– BaseMatrix – defibrinated / delipidated human plasma

Stored at various temperatures: +4°C, –20°C and –80°C

Testing time points: 0, 15, 30, 90, 180, 365 days

DENV-2 preparation (concentration in PCR Detectable units = PDU)

DENV-2 preparation (concentration in PCR Detectable units = PDU)

DENV-1 preparation (concentration in PCR Detectable units = PDU)

DENV-3 preparation (concentration in PCR Detectable units = PDU)

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DENV-3 preparation (concentration in PCR Detectable units = PDU)

DENV-4 preparation (concentration in PCR Detectable units = PDU)

DENV-4 preparation (concentration in PCR Detectable units = PDU)

Next Steps and Future Plans

1. Second round of NAT testing will be performed using stocks diluted in BaseMatrix

2. Perform data analysis on NAT results from second round of testing to further define PDU

3. Initiate contact with WHO collaborating centers to proceed with international evaluation

4. Panels will be made available when adequately standardized and needed